Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers.

Virus-like particles (VLPs) are virus-derived structures made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place in various living or cell-free expression systems after which the viral structures can be assembled and reconstructed. VLPs are gaining in popularity in the field of preventive medicine and to date, a wide range of VLP-based candidate vaccines have been developed for immunization against various infectious agents, the latest of which is the vaccine against SARS-CoV-2, the efficacy of which is being evaluated. VLPs are highly immunogenic and are able to elicit both the antibody- and cell-mediated immune responses by pathways different from those elicited by conventional inactivated viral vaccines. However, there are still many challenges to this surface display system that need to be addressed in the future. VLPs that are classified as subunit vaccines are subdivided into enveloped and non- enveloped subtypes both of which are discussed in this review article. VLPs have also recently received attention for their successful applications in targeted drug delivery and for use in gene therapy. The development of more effective and targeted forms of VLP by modification of the surface of the particles in such a way that they can be introduced into specific cells or tissues or increase their half-life in the host is likely to expand their use in the future. Recent advances in the production and fabrication of VLPs including the exploration of different types of expression systems for their development, as well as their applications as vaccines in the prevention of infectious diseases and cancers resulting from their interaction with, and mechanism of activation of, the humoral and cellular immune systems are discussed in this review.

Correction to: CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.

An amendment to this paper has been published and can be accessed via the original article.

Immunization with oral and parenteral subunit chimeric vaccine candidate confers protection against Necrotic Enteritis in chickens.

Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM. The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.

CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.

Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR's remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR-Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed.

In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens.

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.